1. Field of the Invention
This invention relates to devices and methods for forming gels for electrophoresis and for performing electrophoresis using those gels. More specifically, the invention relates to devices and methods for facilitating electrophoresis in horizontally oriented gels.
2. Related Art
Electrophoretic separation of nucleic acids in agarose gels is an important technique which is commonly used in molecular biology research and genetic diagnosis.
Many devices have been designed to facilitate gel electrophoresis of biologically significant macromolecules. Some of these devices are designed so as to orient the gel vertically. Other devices are designed to orient the gel horizontally.
A vertical orientation for the gel was generally preferred for the electrophoresis of nucleic acids in such applications as nucleic acid sequencing. The disadvantages of devices employing vertical orientation of the gel caused them to be replaced for many other applications by devices in which agarose gels were cast on a horizontal surface so that the electrophoretic separation occurred in a substantially horizontal direction
To cast a horizontal gel, one must seal the ends of a gel casting deck onto which the heated molten gel material is poured. The end sealed casting deck thus provides a shallow cavity in which the molten agarose can cool and gel. Furthermore, to run an agarose gel, it is necessary to have effective and uniform electrical contact between the gel and the electrophoresis buffer at the ends of the gel. Several known devices have been used to cast horizontal agarose gels.
A first approach to casting horizontal gels makes use of "wicks" of agarose cast in chambers at each end of the platform upon which "separation" agarose gel is later cast. The wicks seal in the molten agarose during the casting procedure. The wicks provide an effective electrical connection between the separation gel and buffer reservoirs located on the outside of the respective wicks. An apparatus which typifies this design is disclosed in U.S. Pat. No. 4,234,400 to Kaplan et al.
Another approach adopted by designers of horizontal gel electrophoresis devices employs a removable tray (or deck) upon which the agarose gel could be cast. Certain of these devices cast a gel in an apparatus separate from that used to carry out the actual electrophoresis. Typically, the deck in which the agarose gel is cast consists of two opposing sides and two open ends which, when the gel is being used to electrophoretically separate macromolecules, will be directly exposed to the buffer. Commonly, this deck is situated between two reservoirs which contain a buffered salt solution. Electrodes are installed into the respective reservoirs to provide electrical current from a power supply. The two open ends of the deck allow effective and uniform electrical contact between the gel and the buffer. To cast the gel in the first place, however, it is necessary to seal these open ends of the deck to create a fluid-tight cavity for containing the molten agarose solution before it cools and gels.
To use this gel after hardening, the seals at the ends of the casting deck first have to be removed. A first method used to block the open ends of the gel casting deck during the gel cooling and hardening process consists of sealing the ends of the deck with an adhesive tape. The tape is peeled off before installing the gel in the electrophoresis apparatus. Unfortunately, the removal of the tape is time consuming and requires that the gel be manually picked up. Any such manipulation or other disturbance to the gel is undesirable, since agarose gels are fragile.
A second method of blocking the open ends of the casting deck during the gelation process consists of placing rectangular blocks into slots cut into the side walls of the electrophoresis apparatus. These blocks are thereby positioned against the open ends of the deck, providing a surface which seals the open ends of the deck during the casting. Unfortunately, to achieve a reliably tight seal against the ends of the deck is difficult, since the reliability of the seal in this technique is dependent on the exact positioning of the side wall slots and the exact size and shape of the rectangular blocks. This rectangular block design requires that the device be constructed with very tight manufacturing tolerances which have to be maintained throughout the life of the device. Such tight tolerance requirements cause increased manufacturing costs.
U.S. patent application Ser. No. 081,692 filed Aug. 4, 1987, now U.S. Pat. No. 4,830,725 assigned to a common assignee, discloses a method and apparatus which uses wedges instead of blocks to seal the ends of the deck. Using wedges solves many of the problems associated with blocks and tape.
U.S. Pat. No. 4,473,452 to Cantor et al. discloses an apparatus for and method of electrophoretically separating particles by electric fields which are transverse to each other, which alternate between respective high and low intensities out of phase with each other at a frequency related to the mass of the particles, and which move the particles in an overall direction transverse to the respective directions of the fields.
U.S. patent application Ser. No. 876,523 filed Aug. 13, 1986, now abandoned is directed toward a method and apparatus for manipulating the shape and orientation of electric fields for the purpose of controlling the migration particles in a gel. The invention described in the '523 Application is partly predicated on the discovery that the limitations inherent in the existing electrophoretic separation techniques can be overcome by applying contour-clamped homogeneous or inhomogeneous electric fields. The electric fields are generated by a method in which multiple electrodes are arranged on a closed-contour and clamped to predetermined electric potentials. The '523 Application has greatly enhanced the usefulness of electrophoresis as a research tool. DNA molecules up to or greater than 2 megabases in size can now be separated with high resolution. DNA molecules less than 50 kilobase in size can now be separated without distortion even at high voltage.
Previous electrophoresis techniques only use fields in one direction. Therefore only two opposite sides of the gel needs to be in electrical contact with the buffer. The recent advances in electrophoresis techniques as disclosed by the '523 Application utilize electric fields in more than one direction.
Therefore, when utilizing the electrophoresis techniques taught in the '523 Application, the agarose gel must have direct contact with the electrophoresis buffer on all of its horizontal edges. A simple, economic, repeatable, and convenient means for sealing and unsealing all the sides of gel casting decks and presenting that gel for electrophoresis is therefore desirable in order to practically implement those advances.
One method currently in use for casting gels with all their horizontal edges exposed is to apply adhesive tape to the edges of a rectangular sheet of glass or plexiglass so as to form a shallow pan. The molten gel is then poured into the "pan". After the gel has cooled and solidified, the tape is carefully removed
The above described technique has many shortcomings. It is time consuming. It can easily lead to damaged and unusable gels if the tape is not removed with a large degree of care. Finally, it provides no practical means of transporting the gel about the laboratory after the electrophoresis has taken place without the fear that the slightest mishap will cause the gel to slide off the glass and onto the floor.
Other known techniques for casting gels with all their horizontal edges exposed use the electrophoresis tank itself as part of the casting apparatus. These techniques have the drawback of not allowing multiple gels to be cast in advance and thereby maximize the usage of the electrophoresis equipment. Furthermore, removing the gel from the electrophoresis tank and transporting the gel are often quite difficult. During removal and transport of the gels, they can easily be damaged if not handled with the utmost care. If a gel is damaged, the entire casting and electrophoresis processes may have to be repeated to ensure reliable test results
Another problem present in known electrophoresis devices relates to the difficulty in referencing the "combs" which are used to create "wells" in the agarose gel into which samples are placed. Many known devices are designed so that, when the teeth of the combs are inserted into the molten agarose, the combs rest on surfaces which are physically remote from the casting deck. This remote referencing results in nonuniform and inaccurate separation of the ends of the teeth from the floor of the casting deck due to unreliable fitting of adjacent parts in the apparatus. This nonuniformity and inaccuracy in the placement of the combed teeth results in the creation of wells in the gel which are commensurately nonuniform and inaccurate. At times, the teeth of combs in known devices actually contact the floor of the casting deck, so that there is no agarose seal between the well and the casting deck after the agarose had gelled. The lack of an agarose gel seal sometimes precludes obtaining valuable experimental results.
It is therefore desirable to have a comb reference surface which substantially guarantees the uniformity and accuracy of the depth of wells in agarose gels, and thereby ensures the presence of agarose seals at the bottom of the wells Such a reference surface would reduce the manufacturing tolerances needed to manufacture the apparatus.